Fibrillar type I collagen is the predominant organic component in the mineralized tissues, except tooth enamel, providing a stable template to spatially control the deposition and packing of the mineral crystals. It has become clear that the pattern of collagen post-translational modifications is a critical factor for the formation of functional fibrils that eventually get mineralized. O-glycosylation of collagen is one of such unique modifications occurring at the specific hydroxylysine residues catalyzed sequentially by hydroxylysyl galactosyltransferase (GT) and, then, galactosylhydroxylysyl glucosyltransferase (GGT). Though alterations in collagen glycosylatioon have been implicated in collagen maturation and various bone disorders, the precise mechanisms and the functions of this modification in bone are not well understood. Most recently we have demonstrated that lysyl hydroxylase 3 functions as GGT and, possibly, glycosyltransferase 25 family member 1 (GLT25D1) as GT for bone type I collagen. Based on these and preliminary data, we hypothesized that by manipulating the expression of gene encoding GLT25D1 in osteoblastic cells, under- or overglycosylated type I collagen can be synthesized, and that this system can be used to elucidate the function of glycosylation in collagen maturation and mineralization in bone. To test the hypothesis, the following specific aims are proposed: Aim 1. To generate type I collagen with varied levels of glycosylation in osteoblastic cells and to define its role in collagen maturation and mineralization in vitro, Aim 2. To determine the effects of collagen glycosylation on collagen maturation and mineralization in an in vivo transplantation system. The proposed study should provide insights into the functions of glycosylation in collagen maturation and mineralization, thus, its involvement in bone formation and pathology, and its potential use for the assessment of bone quality. PUBLIC HEALTH RELEVANCE: The quality of fibrillar type I collagen is an important determinant for bone functions and it is established in part by the post-translational modifications. The goal of this study is to elucidate the mechanism and functions of collagen glycosylation in bone, and the results will advance our understanding of bone mineralization and could provide new insights into the mechanisms of bone disorders.